Acute myeloid leukemia (AML), a clonal hematological malignancy, originates from and is sustained by a small population of self-renewing precursor cells - leukemic stem cells (LSCs). This disease is organized by a hierarchy system where the bulk of leukemic cells, i.e. blasts at various stages of maturation, are generated from LSCs through the process known as repopulation/differentiation. Here, leukemic blasts are rapidly expanded and evoke devastating pathological effects in multiple organs. LSCs are also the major source for metastasis, drug resistance, and relapse of the disease. This immortal reservoir of cancer cells display extremely low proliferation rates and likely are not eradicated by current treatments. Clearly, a novel approach focused on the unique properties of LSCs is needed. Our recent studies have established a critical role of PTPMT1, a mitochondrial PTEN-like phosphotidylinositide phosphate phosphatase, in differentiation of embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs). This phosphatase is essential for the metabolic transition from glycolysis to mitochondrial oxidative phosphorylation required for ESC and HSC differentiation, owing to the quickly rising energy demand during this process. PTPMT1 depletion alters mitochondrial aerobic metabolism and causes bioenergetic stress, leading to cell cycle changes and thus a differentiation block in ESCs and HSCs (without affecting cell survival). Intriguingly, PTPMT1 is dispensable for differentiated embryonic fibroblasts and lineage-committed hematopoietic progenitors. These studies led to the identification of a stem cell-specific differentiation checkpoint activated by bioenergetic stress. As LSCs share certain properties with normal HSCs, including metabolic reprogramming during differentiation, we hypothesize that PTPMT1 plays a similarly important role in the progression of LSCs to the blast stage and that LSC differentiation/repopulation capabilities can be blocked via activation of the energetic stress-induced differentiation checkpoint through inhibition of PTPMT1. We plan to test our hypothesis and accomplish the objective of this application by pursuing two aims. 1). To determine the role of PTPMT1 in LSC differentiation/repopulation. 2). To test for the therapeutic effects of a PTPMT1 inhibitor in the AML xenograft model. This application tests a novel idea, i.e. blocking LSC function by inducing metabolic stress, which represents an innovative approach to potentially control AML. In addition, as the PTPMT1 selective inhibitor to be tested is also a known antibiotic, this work may lead to the identificatin of a new therapeutic agent in eliciting a differentiation block in LSCs, thus preventing leukemic blast formation in AML.